Compositions and methods for modifying cell surface glycans

ABSTRACT

Methods and compositions for modifying glycans (e.g., glycans expressed on the surface of live cells or cell particles) are provided herein.

RELATED APPLICATIONS

The present application claims priority to and is a continuation of U.S.Non-provisional application Ser. No. 15/864,975, now abandoned, filed onJan. 8, 2018, which claims priority to and is a continuation of U.S.Non-provisional application Ser. No. 14/487,290, now issued as U.S. Pat.No. 9,914,913, filed on Sep. 16, 2014, which application claims priorityto and is a continuation of U.S. Non-provisional application Ser. No.13/245,129, now issued as U.S. Pat. No. 8,852,935, filed on Sep. 26,2011, which application claims priority to and is a continuation of U.S.Non-provisional application Ser. No. 11/810,256, now issued as U.S. Pat.No. 8,084,236, filed on Jun. 4, 2007, which application claims priorityto and the benefit of U.S. Provisional Application No. 60/810,469, filedJun. 2, 2006, the entire contents of each of which are incorporated byreference in their entirety as if recited in full herein.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The work described herein was funded, in part through grants from theNational Institutes of Health (grants RO1 HL073714 and RO1 HL060528).The United States government may, therefore, have certain rights in theinvention.

FIELD OF THE INVENTION

This invention relates to compositions and methods for modifyingcell-surface glycans on live cells using exogenous glycosyltransferases.Particularly, the composition of methods of the invention preserves theviability and one or more native phenotypic characteristics of thetreated cell.

BACKGROUND OF THE INVENTION

The capacity to direct migration of blood-borne cells to a predeterminedlocation (“homing”) has profound implications for a variety ofphysiologic and pathologic processes. Recruitment of circulating cellsto a specific anatomic site is initiated by discrete adhesiveinteractions between cells in flow and vascular endothelium at thetarget tissue(s). The molecules that mediate these contacts are called“homing receptors”, and, as defined historically, these structures pilottropism of cells in blood to the respective target tissue. At present,only three tissue-specific homing receptors are recognized: L-selectinfor peripheral lymph nodes, α₄β₇ (LPAM-1) for intestines andgut-associated lymphoid tissue, and Cutaneous Lymphocyte Antigen (CLA)for skin (1). Apart from these tissues, it has also been recognized forseveral decades that circulating cells, especially hematopoietic stemcells, navigate effectively to bone marrow (2). However, extensiveinvestigations on this process over several decades have yielded complexand sometimes conflicting results, providing no direct evidence of ahoming receptor uniquely promoting marrow tropism.

From a biophysical perspective, a homing receptor functions as amolecular brake, effecting initial tethering then sustained rollingcontacts of cells in blood flow onto the vascular endothelium atvelocities below that of the prevailing bloodstream (Step 1) (1).Thereafter, a cascade of events ensue, typically potentiated bychemokines, resulting in activation of integrin adhesiveness (Step 2),firm adherence (Step 3) and endothelial transmigration (Step 4) (3).This “multi-step paradigm” holds that tissue-specific migration isregulated by a discrete combination of homing receptor and chemokinereceptor expression on a given circulating cell, allowing forrecognition of a pertinent “traffic signal” displayed by the relevantvascular adhesive ligands and chemokines expressed within targetendothelium in an organ-specific manner. Following engagement of homingreceptor(s) directing trafficking of cells to bone marrow, several linesof evidence indicate that one chemokine in particular, SDF-1, plays anessential role in Step 2-mediated recruitment of cells to this site (2,4, 5).

The most efficient effectors of Step 1 rolling interactions are theselectins (E-, P- and L-selectin) and their ligands (1). As the nameimplies, selectins are lectins that bind to specialized carbohydratedeterminants, consisting of sialofucosylations containing anα(2,3)-linked sialic acid substitution(s) and an α(1,3)-linked fucosemodification(s) prototypically displayed as the tetrasaccharide sialylLewis X (sLe^(x); Neu5Acα2-3Galß1-4[Fucα1-3]GlcNAcß1-)) (1, 6). E- andP-selectin are expressed on vascular endothelium (P-selectin also onplatelets), and L-selectin is expressed on circulating leukocytes (1).E- and P-selectin are typically inducible endothelial membrane moleculesthat are prominently expressed only at sites of tissue injury andinflammation. However, the microvasculature of bone marrowconstitutively expresses these selectins (5, 7), and in vivo studieshave indicated a role for E-selectin in recruitment of circulating cellsto marrow (5, 8). Importantly, SDF-1 is constitutively expressed in highconcentration within the marrow and is co-localized uniquely withE-selectin on the specialized sinusoidal endothelial beds that recruitblood-borne cells to the bone marrow (5).

Two principal ligands for E-selectin have been identified on humanhematopoietic stem/progenitor cells (HSPC), PSGL-1 (9) and a specializedsialofucosylated CD44 glycoform known as Hematopoietic CellE-/L-selectin Ligand (HCELL) (10, 11). CD44 is a rather ubiquitous cellmembrane protein, but the HCELL phenotype is found exclusively on humanHSPCs. In contrast to HCELL's restricted distribution, PSGL-1 is widelyexpressed among hematopoietic progenitors and more mature myeloid andlymphoid cells within the marrow (9). HCELL is operationally defined asCD44 that binds to E-selectin and L-selectin under shear conditions, andis identified by Western blot analysis of cell lysates as a CD44glycoform reactive with E-selectin-Ig chimera (E-Ig) and with mAbHECA452, which recognizes a sialyl Lewis X-like epitope. Like allglycoprotein selectin ligands, HCELL binding to E- and L-selectin iscritically dependent on α(2,3)-sialic acid and α(1,3)-fucosemodifications (10-13). On human HSPCs, HCELL displays the pertinentsialofucosylated selectin binding determinants on N-glycans (10, 12). Invitro assays of E- and L-selectin binding under hemodynamic shear stressindicate that HCELL is the most potent ligand for these moleculesexpressed on any human cell (10, 13). Importantly, though E-selectin isconstitutively expressed on microvascular endothelium of the marrow,this molecule is prominently expressed on endothelial beds at all sitesof tissue injury (e.g., sites of ischemia-reperfusion injury or trauma)or inflammation.

SUMMARY OF THE INVENTION

The invention features compositions and methods for modifying glycansexpressed on the surface of living, e.g. viable cells. The compositionand methods allow modification of cell-surface glycans while preservingcell viability and one or more phenotypic characteristics of the cell.For example, the methods and compositions can be employed to modifyparticular phenotypic characteristics of the cell (such asglycosylation) while preserving one or more other phenotypiccharacteristics (e.g., mutipotency) of the cell.

In one aspect, the invention features a composition for modifying (exvivo or in vitro) a glycan, e.g., a glycan expressed on the surface of acell or a particle or cell fragment (e.g., a mammalian cell or aplatelet or a cell membrane-derived substance/fragment such as aliposome. The composition includes a purified glycosyltransferase (e.g.,a recombinant glycosyltransferase) and a physiologically acceptablesolution, wherein the physiologically acceptable solution is free of oneor more divalent metal co-factors (e.g., the solution is free ofmanganese, magnesium, calcium, zinc, cobalt or nickel). In variousembodiments, the glycosyltransferase is a fucosyltransferase (e.g., analpha 1,3 fucosyltransferase, e.g., an alpha 1,3 fucosyltransferase III,alpha 1,3 fucosyltransferase IV, an alpha 1,3 fucosyltransferase VI, analpha 1,3 fucosyltransferase VII or an alpha 1,3 fucosyltransferase IX),a galactosyltransferase, or a sialyltransferase.

The composition can include more than one glycosyltransferase and/or mayinclude one or more additional agents, such as a donor substrate (e.g.,a sugar). Donor substrates include fucose, galactose, sialic acid, orN-acetyl glucosamine.

The glycosyltransferase has enzymatic activity. Optimally, theglycosyltransferase is capable of transferring 1.0 μmole of sugar perminute at pH 6.5 at 37° C. The composition does not affect integrinadhesion of the cell or cell particle.

The composition can include any physiologically acceptable solution thatlacks divalent metal co-factors. In various embodiments, thephysiologically acceptable solution is buffered. The physiologicallyacceptable solution is, e.g, Hank's Balanced Salt Solution, Dulbecco'sModified Eagle Medium, a Good's buffer (see N. E. Good, G. D. Winget, W.Winter, T N. Conolly, S. Izawa and R. M. M. Singh, Biochemistry 5, 467(1966); N. E. Good, S. Izawa, Methods Enzymol. 24, 62 (1972) such as aHEPES buffer, a 2-Morpholinoethanesulfonic acid (MES) buffer, phosphatebuffered saline (PBS).

In various embodiments, the physiologically acceptable solution is freeof glycerol.

The compositions can be used for modifying a glycan on the surface of acell such as a stem cell (e.g., a mesenchymal stem cell, a hematopoieticstem cell), a progenitor cell (e.g., a neural stem/progenitor cell orpulmonary stem/progenitor cell) or a cell of hematopoietic lineage(e.g., a leukocyte, a lymphocyte), or a cell particle (e.g., a platelet)or a liposome

In another aspect, the invention features a kit for modifying a glycanon the surface of a cell or particle. The kit includes a purifiedglycosyltransferase, and instructions for contacting a cell with theglycosyltransferase in a physiologically acceptable solution which isfree of one or more divalent metal co-factors.

In another aspect, the invention features a method for modifying aglycan on the surface of a cell or particle. The method includescontacting a cell or cell particle with a glycosyltransferase in aphysiologically acceptable solution free of divalent metal co-factorsunder conditions in which the glycosyltransferase has enzymatic activityand the viability of the cell or cell particle population is at least70%, 80%, 90%. 95%, 97%. 98%, 99% or more. Viability is measures a 2, 4,6, 8, 12, 24 hours after contact with the glycosyltransferase.

In various embodiments, the cell or particle is contacted with more thanone glycosyltransferase and its appropriate donor substrate (e.g.sugar). For example, the cell is contacted with two glycosyltransferasessimultaneously, or sequentially, each adding a distinct monosaccharidein appropriate linkage to the (extending) core glycan structure). Themethod is useful, e.g., for modifying glycans on the surface of cells,e.g. stem cells or differentiated cells or cell particles such asplatelets. Cells include for example a mesenchymal cell, hematopoieticstem cells, tissue stem/progenitor cells such as a neural stem cell, amyocyte stem cell, or a pulmonary stem cell, an umbilical cord stemcell, an embryonic stem cell or a leukocyte. The cell or cell particleexpresses CD44, e.g., α (2, 3) sialyated CD44. The cell or cell particledoes not express CD34 or PSGL-1. After modification the cell or cellparticle binds E-selectin and or L-selectin. The modified cell or cellparticles do not bind P-selectin

In various aspects the methods are useful to increase the affinity ofthe cells for a ligand, and/or to increase the in vivoengraftment/homing potential of the cells when administered to asubject, to prevent clearance of administered cells or platelets (extendthe circulatory half-life), or to alter the ability of a platelet toaggregate or to bind to substrates (e.g., endothelium, leukocytes,extracellular matrix, etc.).

Also included in the invention are the cells or cell particles producedby the methods of the invention.

The invention also features methods of increasing engraftment potentialof a cell, treating or alleviating a symptom of an immune disorder,tissue injure or cancer by administering to a subject, e.g. human acomposition comprising the cells of the invention.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 . Human mesenchymal stem cells (MSC) express CD44 and react withSACK-1 mAb, which recognizes a sialic acid-dependent epitope displayedon an N-glycan substitution exclusively carried on a CD44 scaffold. (a)SACK-1 staining of Western blots of untreated (−) orN-Glycosidase-F-treated (+) immunoprecipitated CD44 from KG1a cells (ahuman cell line that natively expresses HCELL) resolved on a reducing4-20% SDS-PAGE gel. (b) (Left panel) Flow cytometry analysis of SACK-1expression on untreated (gray histogram) or sialidase treated (whitehistogram) KG1a cells. (Right panel) SACK-1 staining of Western blots ofuntreated (−) or sialidase-treated (+) immunoprecipitated CD44 from KG1acells resolved on reducing 4-20% SDS-PAGE gel. SACK-1 reactivity ismarkedly diminished following sialidase treatment, as shown by both flowcytometry and Western blot. (c) Flow cytometry analysis of E-selectinligand activity (E-selectin-Ig chimera (E-Ig) binding) and of PSGL-1,CD44, SACK-1, HECA452, KM93 (sLe^(x)), CD11a/CD18 (LFA-1), CD49d/CD29(VLA-4) and CXCR4 expression on MSC. Dotted line is isotype control,black line is specific antibody (or E-Ig chimera); shaded histogram onSACK-1 profile denotes reactivity following sialidase treatment of MSC.Results shown are representative of multiple histograms from MSC derivedfrom multiple marrow donors. Note that human MSC express CD44 and a CD44glycoform displaying SACK-1 determinants, but do not express E-selectinligands (no staining with E-selectin-Ig chimera); they also lack CXCR4and PSGL-1, and also lack the sLe^(x) determinants recognized by KM93and HECA452 mAbs.

FIG. 2 . FTVI treatment of human MSC elaborates sialofucosylations onN-linked glycans of CD44 rendering HCELL expression. (a) Flow cytometryanalysis of HECA452, KM93 (sLe^(x)) and E-Ig reactivity on untreated andFTVI-treated MSC. Dotted line is untreated MSC, black line isFTVI-treated MSC. (b) Western blot analysis of HECA452 (left panel) andof E-Ig (right panel) reactivity of MSC lysates resolved on a reducing4-20% SDS-PAGE. Amounts of lysates in each lane are normalized for cellnumber of untreated and FTVI-treated MSC. Staining with E-Ig wasperformed in the presence (+) or absence (−) of Ca²⁺. Note that FTVItreatment induces HECA452-reactive sialofucosylations and E-Ig bindingselectively on a ˜100 kDa glycoprotein. (c) MSC were treated (+) oruntreated (−) with FTVI. Thereafter, CD44 was immunoprecipitated (usinganti-CD44 mAb Hermes-1) from equivalent cell lysates of FTVI-treated anduntreated cells, and immunoprecipitates were digested with N-glycosidaseF(+) or buffer treated (−). Immunoprecipitates were then resolved byreducing SDS-PAGE (4-20% gradient) and blotted with HECA452, E-Ig oranother anti-human CD44 mAb (2C5). As shown, N-glycosidase F treatmentabrogates HECA452 and E-Ig staining of CD44 from FTVI-treated MSC.Results shown are representative of multiple experiments from MSCderived from several marrow donors.

FIG. 3 . FTVI-treated human MSC display markedly enhancedshear-resistant adhesive interactions with endothelial E-selectin underdefined shear stress conditions. Untreated, FTVI-treated or sialidasedigested FTVI-treated MSC were perfused over IL-1β and TNF-α-stimulatedhuman umbilical vein endothelial cells (HUVEC) at 0.5 dyne/cm². Theaccumulation of relevant MSC was determined at shear stress of 0.5, 1,2, 5, 10, 20 and 30 dyne/cm². In certain instances, EDTA was added tothe assay buffer or HUVEC were pretreated with a function blocking mAbto E-selectin prior to use in adhesion assays. Values are means±SEM (n=4for each group).

FIG. 4 . FTVI-treated, HCELL-expressing human MSC home efficiently tobone marrow in vivo. Montage images of parasagittal region of calvariumassembled from representative experiments of NOD/SCID mice. (a) Allimages shown in this set of panels were obtained at 1 hour afterinfusion of relevant cells: Left panel, untreated MSC; Middle panel,FTVI-treated cells digested with sialidase; Right panel, FTVI-treatedcells. (b) Results shown are from representative images of one mouse at1 hour (left panel) and 24 hours (middle panel) after infusion ofFTVI-treated MSC. Right panel shows high power color image of sinusoidalperivascular region 24 hours after injection of FTVI-treated MSC,revealing extravascular (parenchymal) infiltrates of infusedFTVI-treated MSC: Red speckles are DiD-labeled MSC, green colorhighlights the sinusoidal vessels, visualized by injection offluorescent quantum dots (805 nm).

FIG. 5 is a photograph of a Western Blot showing HECA452 reactivity ofNeural Stem Cells Treated with Fucosyltransferase-VI

FIG. 6 is Flow Cytometric Analysis of HECA452 Expression on Neural StemCells Before and After Fucosyltransferase-VI Treatment.

FIG. 7 Flow Cytometric Analysis of HECA452 Expression on Pulmonary StemCells Before and After Fucosyltransferase-VI Treatment

DETAILED DESCRIPTION

The invention is based in part on the surprising discovery thatglycosyltransferases retain enzymatic activity in the absence ofdivalent metal co-factors (e.g. divalent cations such as manganese,magnesium, calcium, zinc, cobalt or nickel) and stabilizers such asglycerol. Previously, divalent metal co-factors had been deemed criticalfor enzymatic activity. The glycosyltransferase compositions accordingto the invention are particularly useful in modification of glycans onlive cells. Previous attempts to modify glycan structures on live cellsresulted in cell death and phenotypic changes to the cell due the toxiceffects of the metal co-factors and enzyme stabilizers such as glycerol.For ex vivo custom engineering of live cell surface glycans usingglycosyltransferases, it is essential that the target cells remainviable and phenotypically conserved following treatment(s). Inapplications utilizing stem cells, it is also important to analyzewhether differentiation along characteristic lineages is affected byenzymatic treatment.

Beyond their recognized effects on cell viability (20), divalent metalco-factors such as Mn⁺⁺ itself triggers signal transduction (21) andactivates integrin-adhesiveness (e.g., for VLA-4) at levels well belowthose employed in forced glycosylation (e.g., fucosylation) (22, 23).These Mn⁺⁺ effects are confounders to the effect(s) of glycosylation oncellular trafficking, as the resulting integrin-mediated firm adhesionwould be manifest rampantly at endothelial beds and within tissueparenchyma expressing relevant ligands.

To address these concerns, a new method for high titerfucosyltransferase production in a Pichia Pastoris system was developed.Additionally, the fucosyltransferase was stabilized in a buffer (e.g.,HBSS) specifically chosen to minimize cell toxicity. Furthermore, enzymeconditions were refined to utilize physiologic buffers in the couplingreaction without input of divalent metal co-factors (e.g., without inputof Mn⁺⁺ ions)

These experimental modifications resulted in high efficiencyfucosylation of CD44 on MSCs with 100% cell viability followingenzymatic treatment. Importantly, kinetic analysis following forcedfucosylation showed that cell viability in vitro was retainedindefinitely after treatment in all MSC, yet HCELL expression wastransient: HCELL levels were stable for 24 hours and declined steadilythereafter to baseline (no HCELL) by 96 hours, presumably reflectingcell turnover of membrane CD44. Importantly, there was no effect on MSCdifferentiation into various lineages following FTVI treatments, assayeddaily for up to 2 weeks following treatment. Thus, FTVI treatment had noapparent effect on the phenotype of MSC, with exception only of HCELLexpression (FIG. 2 c ). In contrast, FTVI treatment of MSC fromcommercial available FTVI (e.g., compositions containing Mn⁺⁺ andglycerol; Calbiochem) while enhancing HCELL expression cell viabilitywas compromised following these FTVI treatments, with >95% of cellsdying within 8 hours of modification. This loss of viability wasattributed to exposure to stabilizers (e.g., glycerol) in the commercialenzyme formulations and to exposure to high levels of Mn⁺⁺ (10 mM) usedin the enzymatic reaction. Accordingly, the compositions of theinvention now make it feasible to ex-vivo engineer glycans on a surfaceof a viable cell to produce a therapeutic product that is suitable forin vivo administration to a human.

Following forced fucosylation, and despite absence of surface CXCR4expression, intravenously infused MSC homed robustly to bone marrow, atissue constitutively expressing vascular E-selectin. These findingsestablish HCELL as a human bone marrow homing receptor, provide directevidence that CXCR4 engagement is not obligatory for marrow trafficking,and present new perspectives on the multi-step paradigm.

The finding that enforced HCELL expression confers marrow tropism, andthat its functional inactivation by sialidase treatment specificallyreverses this effect, defines this CD44 glycoform as a “bone marrowhoming receptor”. As such, the ability to custom-modify HCELL expressionex vivo may be useful for improving engraftment of HSPCs in clinicaltransplantation, or for use of MSC in cell-based therapy (e.g., for bonediseases). More generally, the data suggest that enforcing cellularHCELL expression may promote systemic delivery to tissues whoseendothelial beds express E-selectin. The high specificity and efficiencyof this rather subtle fucose modification ofα(2,3)-sialylated-glycoforms of CD44 thus provides guiding principlesand technologies for strategies to selectively upregulate HCELLexpression for adoptive cellular therapeutics. The facility with whichthis can be accomplished suggests that rapid translation of thisapproach to patients should be straightforward. Because E-selectin isdisplayed prominently at sites of inflammation and ischemia in affectedtissues of primates (29, 30), modulation of HCELL expression could leadto directed migration and infiltration of progenitor/stem cells atinjured/damaged tissue(s) for regenerative therapeutics. Beyondimplications in stem cell-based therapies, these findings also testingof how upregulated E-selectin ligand activity on other cells, such asimmunologic effector and regulatory cells, may be harnessed to achievetargeted cell migration in a variety of physiologic and pathologicprocesses, including immune diseases, infectious diseases, and cancertherapeutics.

Compositions

The invention provides compositions for ex vivo modification of cellsurface glycans on a viable cell or cell particle. The compositionsinclude a purified glycosyltransferase polypeptide and a physiologicallyacceptable solution free of divalent metal co-factors. The compositionis free of stabilizer compounds such as for example, glycerol.Glycosyltransferase include for example, fucosyltransferase,galactosyltransferase, sialytransferase andN-acetylglucosaminotransferase. The fucosyltransferase is an alpha 1,3fucosyltransferase such as an alpha 1,3 fucosyltransferase III, alpha1,3 fucosyltransferase IV, an alpha 1,3 fucosyltransferase VI, an alpha1,3 fucosyltransferase VII or an alpha 1,3 fucosyltransferase IX)

Optionally, the composition further includes a sugar donor suitable forthe specific glycosyltransferase. For example, when theglycoslytransferase is a fucosyltransferase, the donor is GDP-fucose.Whereas, when the glycosyltransferase is a siayltransferase, the donoris CMP-sialic acid. One skilled in the art would recognize suitablesugar donors.

The glycosyltransferases are biologically active. By biologically activeis meant that the glycosyltransferases are capable of transferring asugar molecule from a donor to acceptor. For example, theglycosyltransferase is capable of transferring 0.1, 0.2, 0.3, 0.4, 0.5,1.0, 1.5, 2.0, 2.5, 5, 10 or more μmoles of sugar per minute at pH 6.5at 37° C.

Physiologically acceptable solution is any solution that does not causecell damage, e.g. death. For example, the viability of the cell or cellparticle is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or moreafter treatment with the compositions of the invention. Suitablephysiologically acceptable solutions include for example, Hank'sBalanced Salt Solution (HBSS), Dulbecco's Modified Eagle Medium (DMEM),a Good's buffer (see N. E. Good, G. D. Winget, W. Winter, T N. Conolly,S. Izawa and R. M. M. Singh, Biochemistry 5, 467 (1966); N. E. Good, S.Izawa, Methods Enzymol. 24, 62 (1972) such as a HEPES buffer, a2-Morpholinoethanesulfonic acid (MES) buffer, or phosphate bufferedsaline (PBS).

Therapeutic Methods

The compositions of the invention, due to their low toxicity on viablecells and high enzymatic activity are useful for the ex vivo or in vitromodification of glycan on the surface of cells or cell particles.Moreover, the modified cells and particles produced using thecompositions and methods of the invention are useful in therapeuticsettings to achieve targeted cell migration in a variety of physiologicand pathologic processes, including bone disease, immune diseases,infectious diseases, and cancer therapeutics. The Federal DrugAdministration imposes (FDA) imposes rigid requirements on all finalcell products for human administration. Specifically, the FDA requires aminimum cell viability of 70%, and any process should consistentlyexceed this minimum requirement. Unlike previous described methods of exvivo or in vitro modification of glycan on the surface of cells whichutilized glycosyltransferases compositions, containing divalent metalco-factors and stabilizers such as glycerol (which resulted insignificant cell death), the methods described herein produce a cellbased product that meets or exceeds the FDA requirements.

More specifically, the glycan engineering of the cell surface will drivehoming of cells to any site where E-selectin is expressed. Inparticular, since CD44 is a ubiquitously expressed cell membrane proteinand is displayed on stem/progenitor cell populations of both “adult” andembryonic types, the capacity to modify glycosylation of this protein byex vivo glycan engineering to create the HCELL (CD44 glycoform)phenotype will drive migration of intravascularly injected (adoptivelytransferred) cells in vivo to marrow or to any tissue/organ site whereE-selectin is expressed.

Glycans are modified on the surface of a cell or cell particle (e.g.platelet or liposome) by contacting a population of cells with one ormore glycosyltransferase compositions according to the invention. Thecells are contacted with the glycosyltransferase composition underconditions in which the glycosyltransferase has enzymatic activity.Glycan modification according to the invention results in cells thathave at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more viability.Viability is determined by methods known in the art such as trypan blueexclusion. Viability is measured 1 hr, 2 hr, 4 hr, 18 hr, 12 hr 24 hr ormore after treatment. The phenotype of the cells (other than the glycanmodification) is preserved after treatment. By preserved phenotype it ismeant the cell maintains its native function and or activity. Forexample, if the cell is a stem cell it retains its pluripotency.

After modification, the cell or cell particle binds E-selectin and orL-selectin. In various aspects, the modified cell does not bindP-selectin. Preferably, after modification the cells express thesialofucosylated CD44 glycoform known as Hematopoietic CellE-/L-selectin Ligand (HCELL). After modification, the cell or cellparticle is capable of homing in-vivo to the bone marrow and or sites ofischemia or inflammation.

The cell or cell particle is any cell in which cell surface glycanmodification is desired. The cell is a stem cell (i.e., multipotent) ora differentiated cell. Stem cells include for example a hematopoieticstem cell, a mesechymal stem cell, a tissue stem/progenitor cell (e.g.,a neural stem cell, myocyte stem cell or pulmonary stem cell), anumbilical cord stem cell, or an embryonic stem cell. Differentiatedcells includes hematopoietic-lineage cells such as a leukocyte, e.g., alymphocyte. The lymphocyte can be a B-lymphocyte or T-lymphocyte, or asubset of T lymphocytes, e.g., a “regulatory” lymphocyte(CD4⁺/CD25⁺/FOXP3⁺).

The cell or cell particle expresses CD44. The CD44 is notsialofucosylated. Alternatively the CD44 is alpha (2,3)-sialylated andlacks relevant fucosylations rendering the HCELL phenotype. Enforcedglycosylation of CD44 to render HCELL is useful in improving engraftmentof hematopoietic stem/progenitor cells (HSPCs) in clinical hematopoieticstem cell transplantation, or for use of MSC in cell-based therapy(e.g., for bone diseases). More generally, the data suggest thatenforcing cellular HCELL expression may promote systemic delivery ofHCELL-bearing cells to tissues whose endothelial beds expressE-selectin.

In various aspects cell does not express PSGL-1, CD34 or both.

The modified cells of the invention because of their increased homingcapabilities are useful for example in for improving engraftment ofHSPCs in clinical transplantation, for use of MSC in cell-based therapy(e.g., for bone diseases) or directing migration and infiltration ofprogenitor/stem cells at injured/damaged tissue(s) for regenerativetherapeutics.

For example, the composition are useful for treating a variety ofdiseases and disorders such as ischemic conditions (e.g., limb ischemia,congestive heart failure, cardiac ischemia, kidney ischemia and ESRD,stroke, and ischemia of the eye), conditions requiring organ or tissueregeneration (e.g., regeneration of liver, pancreas, lung, salivarygland, blood vessel, bone, skin, cartilage, tendon, ligament, brain,hair, kidney, muscle, cardiac muscle, nerve, and limb), inflammatorydiseases (e.g., heart disease, diabetes, spinal cord injury, rheumatoidarthritis, osteo-arthritis, inflammation due to hip replacement orrevision, Crohn's disease, and graft versus host disease) auto-immunediseases (e.g., type 1 diabetes, psoriasis, systemic lupus, and multiplesclerosis), a degenerative disease, a congenital disease hematologicdisorders such as anemia, neutropenia, thrombicytosis,myeloproliferative disorders or hematologic neoplasms and cancer such asleukemia.

Diseases and disorders are treated or a symptom is alleviated byadministering to a subject in need thereof a cell composition producedby the methods of the invention. The cell compositions are administeredallogeneically or autogeneically.

Example 1: General Methods

Reagents: The following antibodies were from BD Pharmingen: functionblocking murine anti-human E-selectin (68-5411; IgG₁), rat anti-humanCLA (HECA-452; IgM), murine anti-human PSGL-1 (KPL-1; IgG₁), purifiedand FITC-conjugated murine anti-human L-selectin (DREG-56; IgG₁), murineanti-human CXCR4 (12G5; IgG_(2a)), FITC-conjugated murine anti-humanCD18 (L130; IgG₁), murine anti-human CD29 (MAR4; IgG₁), PE-conjugatedmurine anti-human CD49d (9F10; IgG₁), mouse IgG₁,κ isotype, mouseIgG_(2a) isotype, mouse IgM isotype, rat IgG isotype and rat IgMisotype. Rat anti-human CD44 (Hermes-1; IgG_(2a)) was a gift of Dr.Brenda Sandmaier (Fred Hutchinson Cancer Research Center; Seattle,Wash.). Recombinant murine E-selectin/human Ig chimera (E-Ig) and murineanti-human CD44 (2C5; IgG_(2a)) were from R&D Systems. Murine anti-humansLe^(x) (KM93; IgM) was from Calbiochem. FITC-conjugated murineanti-human CD11a (25.3; IgG₁), PE-conjugated mouse IgG₁,κ isotype andFITC-conjugated mouse IgM isotype were from Coulter-Immunotech.FITC-conjugated goat anti-rat IgM, FITC-conjugated goat anti-mouse IgG,FITC-conjugated goat anti-mouse IgM, PE-conjugated strepavidin, alkalinephosphatase (AP)-conjugated anti-rat IgM, anti-mouse Ig, and anti-humanIg were from Southern Biotechnology Associates. V. Cholerae sialidasewas from Roche.

Human cells: Bone marrow (BM) cells were obtained from harvest filtersof healthy individuals donating bone marrow for hematopoietic stem celltransplantation at the Brigham & Women's Hospital/Dana Farber CancerInstitute and Massachusetts General Hospital. BM mononuclear cells(BMMNCs) were collected by Ficoll-Paque density gradient centrifugation.Human cells were used in accordance with the protocols approved by theHuman Experimentation and Ethics Committees of Partners Cancer CareInstitutions [Massachusetts General Hospital, Brigham & Women's Hospitaland Dana Farber Cancer Institute (Boston, Mass.)]. Human umbilical veinendothelial cells (HUVEC) were obtained from the tissue culture corefacility at Brigham & Women's Hospital's Pathology Department and weremaintained in M199 supplemented with 15% FBS, 5 units/ml heparin, 50μg/ml endothelial growth factor and 1% penicillin/streptomycin. Tostimulate expression of E-selectin, confluent monolayers of HUVEC werepre-treated with 1 ng/ml IL-1β(Research Diagnostics, Inc; Concord,Mass.) and 10 ng/ml TNF-α (Research Diagnostics, Inc.) for 4-6 hrs priorto use in the adhesion studies.

MSC culture: MSC were maintained in a humidified incubator at 37° C. inan atmosphere of 95% air, 5% CO₂ (as per (15)) or in 3% O₂, 5% CO₂, 92%N₂ (as per (16), “MIAMI” cells). For culture of either type of MSC,BMMNCs were plated initially at a density of 2×10⁵/cm² in DMEM-lowglucose medium supplemented with 10% fetal bovine serum (FBS) fromselected lots. After several days, non-adherent cells were removed andadherent cells were harvested by treatment with 0.05% trypsin/0.5 mMEDTA/HBSS (Invitrogen Corp.) and replated at a density of 50 cells/cm².Medium was replaced at 48 to 72 hours and every third or fourth daythereafter. Cells were replated when density approached 40% confluence.For all experiments, MSC were used within the first 3 passages, andharvested by treatment with 0.05% trypsin/0.5 mM EDTA/HBSS for less than3 minutes at 37° C.

Generation of SACK-1 mAb: HCELL was isolated from KG1a cells byimmunoaffinity chromatography of cell lysates using anti-CD44 mAb.BALB/c mice were injected with pure HCELL in complete Freund's adjuvant(1:1 emulsion), splitting inoculum 50:50 between skin andintraperitoneal sites. Boosting was performed 2 weeks later with pureHCELL, diluted 1:1 in incomplete Freund's adjuvant and injectedintraperitoneally. 10-14 days later, mice were boosted by IV injectionof 5 μg HCELL, then spleens were harvested 3 days following IV boost.Splenocytes were fused with NSO myeloma cells. Screening of hybridomasupernatants was initially performed by flow cytometry, againsthematopoietic cell lines KG1a (CD44+/HCELL+/HECA452+), HL60(CD44+/HCELL−/HECA452+), RPMI8402 (CD44+/HCELL−/HECA452−), JURKAT andK562 (both of which are CD44−/HCELL−/HECA452−). SACK-1 mAb wasidentified as “CD44-specific, carbohydrate-specific”, by reactivity toKG1a but not to CD44− cell lines, in conjunction with Western blotevidence of mono-specificity for CD44 expressed on KG1a cells, sensitiveto digestion with N-glycosidase F (New England Biolabs; N-glycoidase Fdigestion performed as previously described (10, 12)).

Flow cytometry: Aliquots of cells (2×10⁵ cells) were washed with PBS/2%FBS and incubated with primary mAbs or with isotype control mAbs (eitherunconjugated or fluorochrome conjugated). The cells were washed inPBS/2% FBS and, for indirect immunofluorescence, incubated withappropriate secondary fluorochrome-conjugated anti-isotype antibodies.After washing cells, FITC or PE fluorescence intensity was determinedusing a Cytomics FC 500 MPL flow cytometer (Beckman Coulter Inc.,Fullerton, Calif.).

Recombinant expression and formulation of humanα(1,3)-fucosyltransferase VI: Pichia pastoris KM 71 (arg4his4aox1:ARG4)host strain containing the human α(1,3)-fucosyltransferase VI (FTVI)gene and the N-terminal signal sequence of S. cerevisiae α-factor wereused for stable expression and secretion of highly activeα(1,3)-fucosyltransferase VI into the medium using online methanolsensing (sterilizable methanol sensor by Raven Biotech, Vancouver,Canada) and regulation of methanol addition by Alitea-pumps (Alitea A.B., Stockholm, Sweden). After the end of fermentation, the broth wascooled down to 10° C. and the Pichia cells were separated by aPellicon-microfiltration system with 0.2 μm membranes and, subsequently,the final formulation was achieved by buffer exchange with HBSS using aPellicon-ultrafiltration system with 10 kD-UF-membranes (regeneratedcellulose).

Recombinant expression and formulation of human sialytransferase: Pichiapastoris KM 71 (arg4his4aox1:ARG4) host strain containing the humansialytransferase gene and the N-terminal signal sequence of S.cerevisiae α-factor were used for stable expression and secretion ofhighly active sialytransferase into the medium using online methanolsensing (sterilizable methanol sensor by Raven Biotech, Vancouver,Canada) and regulation of methanol addition by Alitea-pumps (Alitea A.B., Stockholm, Sweden). After the end of fermentation, the broth wascooled down to 10° C. and the Pichia cells were separated by aPellicon-microfiltration system with 0.2 μM membranes and, subsequently,the final formulation was achieved by buffer exchange with HBSS using aPellicon-ultrafiltration system with 10 kD-UF-membranes (regeneratedcellulose).

Recombinant expression and formulation of human glycosytransferase:Pichia pastoris KM 71 (arg4his4aox1:ARG4) host strain containing thehuman glycosytransferase gene and the N-terminal signal sequence of S.cerevisiae α-factor is used for stable expression and secretion ofhighly active glycosytransferase into the medium using online methanolsensing (sterilizable methanol sensor by Raven Biotech, Vancouver,Canada) and regulation of methanol addition by Alitea-pumps (Alitea A.B., Stockholm, Sweden). After the end of fermentation, the broth iscooled down to 10° C. and the Pichia cells were separated by aPellicon-microfiltration system with 0.2 μm membranes and, subsequently,the final formulation is achieved by buffer exchange with HBSS using aPellicon-ultrafiltration system with 10 kD-UF-membranes (regeneratedcellulose).

Recombinant expression and formulation of humanN-acetylglucosaminotransferase: Pichia pastoris KM 71(arg4his4aox1:ARG4) host strain containing the humanN-acetylglucosaminotransferase gene and the N-terminal signal sequenceof S. cerevisiae α-factor is used for stable expression and secretion ofhighly active N-acetylglucosaminotransferase into the medium usingonline methanol sensing (sterilizable methanol sensor by Raven Biotech,Vancouver, Canada) and regulation of methanol addition by Alitea-pumps(Alitea A. B., Stockholm, Sweden). After the end of fermentation, thebroth is cooled down to 10° C. and the Pichia cells are separated by aPellicon-microfiltration system with 0.2 μm membranes and, subsequently,the final formulation is achieved by buffer exchange with HBSS using aPellicon-ultrafiltration system with 10 kD-UF-membranes (regeneratedcellulose).

FTVI and Sialidase treatment: MSC either in confluent monolayer or insuspension were treated with 60 mU/mL FTVI in HBSS containing 20 mMHEPES, 0.1% human serum albumin and 1 mM guanosine diphosphate(GDP)-fucose for 40 min. at 37° C. After the incubation, MSC were washedwith HBSS containing 0.2% BSA and 20 mM HEPES. Untreated andFTVI-treated MSC were then used for experiments. In some experiments,MSC were first treated with FTVI and then subjected to sialidasetreatment (100 mU/ml V. Cholerae Sialidase, 1 hour, 37° C.)(“FTVI-Sialidase MSC”). Efficacy of sialidase treatment was confirmed ineach case by loss of reactivity to KM93 and HECA452 by flow cytometry.

Sialytransferase treatment: Cells are treated with 60 mU/mL ofN-sialyltransferase, 1 mM CMP-sialic acid or treated with buffer alone(HBSS, 0.1% human serum albumin) for 1 hour at 37° C. After theincubation, the cells are washed with HBSS containing 0.2% BSA and 20 mMHEPES.

Galactosyltransferase treatment: Cells are treated with 60 mU/mL ofGalactosyltransferas, 1 mM UDP-galactose or treated with buffer alone(HBSS, 0.1% human serum albumin) for 1 hour at 37° C. After theincubation, the cells are washed with HBSS containing 0.2% BSA and 20 mMHEPES.

N-acetylglucosaminotransferase treatment: Cells are treated with 60mU/mL of N-acetylglucosaminotransferase, 1 mM UDP-N-acetylglucosamine ortreated with buffer alone (HBSS, 0.1% human serum albumin) for 1 hour at37° C. After the incubation, the cells are washed with HBSS containing0.2% BSA and 20 mM HEPES.

Western blot analysis: Untreated and FTVI treated MSC were lysed using2% NP-40 in Buffer A (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 20μg/ml PMSF, 0.02% sodium azide; and protease inhibitor cocktail tablet(Roche Molecular Biochemicals)). Western blots of quantified proteinlysates or of immunoprecipitated protein were performed under reducingconditions as described previously (10).

Immunoprecipitation studies: Cell lysates of untreated or FTVI treatedMSC were incubated with immunoprecipitating antibodies or withappropriate isotype controls and then incubated with Protein G-agarose.Immunoprecipitates were washed extensively using Buffer A containing 2%NP-40, 1% SDS. In some experiments, immunoprecipitates were treated withN-glycosidase F (New England Biolabs) as previously described (10, 12)).For Western blot analysis, all immunoprecipitates were diluted inreducing sample buffer, boiled, then subjected to SDS-PAGE, transferredto PVDF membrane, and immunostained with HECA-452, E-Ig, SACK-1 or 2C5(10).

Parallel plate flow chamber adhesion assay: E-selectin binding capacityof untreated and FTVI-treated MSC was evaluated using a parallel plateflow chamber (Glycotech; Gaithersburg, Md.). MSC (0.5×10⁶ cells/ml,suspended in HBSS/10 mM HEPES/2 mM CaCl₂ solution) were drawn overconfluent HUVEC monolayers. Initially, the MSC were allowed to contactthe HUVEC monolayer at a shear stress of 0.5 dyne/cm², subsequently theflow rate was adjusted to exert shear stress ranging from 0.5 to 30dynes/cm². The number of untreated or FTVI-treated MSC adherent to theHUVEC monolayer was quantified in the final 15 sec interval at shearstress of 0.5, 1, 2, 5, 10, 20 and 30 dyne/cm². Each assay was performedat least 3 times and the values averaged. Control assays were performedby adding 5 mM EDTA to the assay buffer to chelate Ca²⁺ required forselectin binding or treating HUVEC with function-blocking anti-humanE-selectin mAb (68-5411) at 37° C. for 15 min. prior to use in adhesionassays.

In vivo horning: All studies were performed in accordance with NIHguidelines for the care and use of animals and under approval of theInstitutional Animal Care and Use Committees of the MassachusettsGeneral Hospital and the Harvard Medical School. For intravitalmicroscopy, NOD/SCID mice were anesthetized and a small incision wasmade in the scalp to expose the underline dorsal skull surface aspreviously described (5). Experiments were performed on the same dayusing littermates to analyze each of four groups of MSC (n=4 for eachgroup): (1) FTVI treated (as above) MSC; (2) Buffer-treated MSC; (3)FTVI-treated MSC digested with sialidase (100 MU/Ml V. CholeraSialidase, 37° C., 1 hour); and (4) untreated MSC. Cells were stainedwith the fluorescent lipophilic tracer dye DiD (10 μM, 37° C., 30 min;Molecular Probes) and infused into tail vein of NOD/SCID mice. Theinteractions of MSC with bone marrow microvascular endothelial cellswithin the parasagittal region were monitored and imaged at differenttime points after injection by in vivo confocal microscopy usingprogressive scanning and optical sectioning combined with video-rateimaging as previously described (5). For delineation of bone marrowvasculature, long-circulating fluorescent quantum dots (Qtracker 800,Invitrogen) were injected systemically just prior to imaging. Stocksolution of Qtracker 800 (2 μM) was diluted 1:4 (50 μL mixed in 150 μLPBS1×) and injected into anesthetized mouse via tail vein. In vivoconfocal microscopy of the mouse skull bone marrow was performed aspreviously described (5). DiD-labeled cells were excited with asolid-state 633 nm laser and imaged with a 45 nm bandpass filtercentered at 695 nm, while quantum dots were excited with a solid-stateNd:YAG laser at 532 nm and imaged with a 770 nm longpass filter.

Example 2: Human Mesenchymal Stem Cells Express N-Linked, SialylatedGlycoforms of CD44 and do not Bind Selectins

Bone marrow contains two populations of stem cells, hematopoietic stemcells and mesenchymal stem cells (MSC). MSC represent a small populationof cells present within normal marrow, but they can be isolated andexpanded in culture. MSC characteristically express CD44 and severalother adhesion molecules found on hematopoietic cells (14). However, itis unknown whether these primitive non-hematopoietic cells express anyselectin ligands. This paucity of data, and the finding that HCELL isexpressed among only the earliest hematopoietic cells (CD34+/lin− cells)(10, 11), prompted us to examine whether MSC display similarcarbohydrate modifications on CD44 that could bind selectins.

MSC were cultured from human bone marrow as per two established,published protocols (15, 16). The MSC derived using both methods werecapable of multipotential differentiation toward adipocyte, osteocyteand fibroblast differentiation, as previously described (15, 16).Regardless of protocol, MSC displayed no significant differences in anyof the measured cell surface markers or in their response to enzymatictreatments. By flow cytometry (FIG. 1 c ), MSC lacked expression ofPSGL-1 or of sialofucosylated determinants that could serve as selectinligand(s): notably, the cells were devoid of reactivity to mAb KM93 orHECA452 (each of which identify sialyl Lewis X) and to E-Ig by both flowcytometry and Western blot (FIGS. 1 c, 2 a and 2 b ). Additionally, bothtypes of MSC lacked LFA-1 (CD11a/CD18) but expressed another integrin,VLA-4 (CD49d/CD29) (FIG. 1 c ). VLA-4 can mediate rolling interactionsand firm adherence on vascular endothelium (17), but both of theseadhesive functions require “inside-out” activation usually mediated bythe SDF-1/CXCR4 pathway (18). Importantly, analysis of immunofluoresencestaining on adherent MSC on plates and by flow cytometry showed noexpression of CXCR4 (FIG. 1 c ) and, predictably, MSC did not migrate inresponse to SDF-1 in either static or flow-based assays (not shown).

CD44 expression was high among all MSC isolated from numerous donors(FIG. 1 c ). Conspicuously, SACK-1 reactivity was also high on all MSCfrom all donors (FIG. 1 c ), indicating that these cells uniformlyexpressed N-linked, sialylated glycoforms of CD44. The native absence ofselectin ligands but presence of a sialylated CD44 acceptor made MSC anideal cell type to examine how HCELL expression affects cellulartrafficking to bone marrow.

Example 3: Ex Vivo Fucosylation of Mesenchymal Stem Cells Results inHCELL Expression

To enforce HCELL expression, MSC were treated ex vivo with anα(1,3)-fucosyltransferase, fucosyltransferase VI (FTVI). In all MSCcultured from all donors, forced fucosylation resulted in profoundstaining with mAb HECA452 and KM93, consistent with expression of sialylLewis X epitopes (FIG. 2 a ). Western blot of cell lysates and ofimmunoprecipitated CD44 from FTVI-treated MSC revealed that the onlyglycoprotein bearing requisite sialofucosylations recognized by HECA452was CD44 (FIGS. 2 b and 2 c ). Moreover, fucosylated MSC bound E-Ig byflow cytometry, and Western blot analysis of cell lysates showed thatthe only glycoprotein supporting E-Ig binding was CD44 (FIG. 2 ). Therelevant sialofucosylations of HCELL were displayed on N-glycans, asshown by abrogation of E-Ig binding following digestion withN-glycosidase F (FIG. 2 c ).

To analyze the E-selectin ligand activity of FTVI-treated MSC underphysiologic blood flow conditions, parallel plate flow chamber studieswere performed using human umbilical vein endothelial cells (HUVEC)stimulated by cytokines to express E-selectin. As shown in FIG. 3 ,FTVI-treated MSC showed profound E-selectin ligand activity, which wascompletely abrogated in the presence of EDTA and by treatment of MSCwith sialidase. Consistent with prior studies of cells nativelyexpressing HCELL, robust shear-resistant interactions were observedwithin usual post-capillary venular shear levels (1-4 dynes/cm²), andpersisted at upwards of 20 dyne/cm², well outside the range where PSGL-1can support E-selectin binding (10). These data indicated that the HCELLcreated by fucosylation of MSC surfaces was functionally similar to thatdisplayed natively on the surface of KG1a cells and human hematopoieticprogenitor cells (10, 11).

Example 4: HCELL Expression Conferred Enhanced Homing of MSC to BoneMarrow in Vivo

To determine whether HCELL expression conferred enhanced homing of MSCto bone marrow in vivo, we employed dynamic real-time confocalmicroscopy to visualize marrow sinusoidal vessels in the calvarium oflive immunodeficient mouse (NOD/SCID) hosts (5). Four groups of cellswere injected into tail vein of respective hosts: (1) FTVI-treated MSC,(2) FTVI-treated MSC digested with sialidase (“FTVI-Sialidase MSC”), (3)buffer-treated MSC, and (4) untreated MSC. In vivo microscopy studiesshowed that FTVI-treated, HCELL-expressing MSC rolled directly on marrowsinusoidal vessels, and infiltrated the marrow parenchyma rapidly,within hours of infusion (FIG. 4 ). In contrast, untreated MSC andbuffer-treated MSC showed minimal binding interactions with sinusoidalendothelium and displayed only modest infiltrates, whereasFTVI-Sialidase MSC typically showed even lower levels of endothelialinteractions and marrow infiltrates (FIG. 4 ). The latter findinghighlights the critical role of HCELL in homing, and also indicates thatthe marrow tropism following FTVI treatment was not a result offucosylation per se or of indirect effects on other adhesion molecules,but is a consequence solely of the induced selectin ligand activity,requiring concomitant expression of α(2,3)-sialic acid and α(1,3)-fucosemodifications. Images obtained with simultaneous staining of MSC andblood vessels clearly show that HCELL+MSC infiltrated the marrowparenchyma (FIG. 4 ). The observed marrow infiltrates are striking giventhat studies herein were performed without injury induction, such as byuse of radiation or other preparative manipulations of recipient animalsthat markedly augment expression of sinusoidal ligands promoting marrowtrafficking (27). Collectively, these data provide definitive evidencethat HCELL expression directly enhances homing of MSC to bone marrow.

In the canonical multi-step paradigm, homing receptor-mediated rollinginteractions on the endothelium facilitates exposure to chemokinespresumed critical for G-protein-coupled upregulation of integrinadhesiveness with resulting firm adhesion followed by transmigration(3). Notably, the MSC used here did not bear CXCR4 or undergo chemotaxisto SDF-1, the principal chemokine regulating bone marrow homing (4, 5).Thus, the capacity of these cells to infiltrate marrow shows that CXCR4engagement is not compulsory for marrow trafficking. However, Step 1interactions are indispensable for cell trafficking to any tissue, and,as shown here, augmentation of E-selectin ligand activity promotesmarrow homing. Viewed more broadly, our findings are consistent with agrowing body of experimental evidence indicating that engagement ofhoming receptors may be sufficient alone (i.e., absent chemokinesignaling) to induce integrin adhesiveneness, with accompanying firmadherence and trans-endothelial migration (1). Notably, it has beenfound that ligation of CD44 itself on lymphocytes results in direct,synergistic upregulation of VLA-4 adhesiveness, leading totransmigration without chemokine involvement (28). Though future studieswill be needed to determine whether this axis operates in other celltypes, the fact that MSC characteristically express VLA-4 (FIG. 1 c )raises this possibility.

Example 5: In Vivo Fucosylation of Neural Stem Cells

Neural stem cells were treated with 60 mU/mL of FT-VI, 1 mM GDP-fucoseor treated with buffer alone (HBSS, 0.1% human serum albumin) for 1 hourat 37° C. Cells were lysed in a buffer containing 2% NP-40. Proteinswere separated on a 4-20% Tris-HCl gradient gel in denaturing conditionsand transferred to a PVDF membrane. Membrane was immunoblotted withHECA452 antibody. Resulting blot shows the expression of HECA452reactive epitopes on a number of proteins after forced fucosylation.FT-VI-treated neural stem cells were also analyzed for HECA452reactivity using flow cytometry. FT-VI-Cells were incubated with 10ug/mL of HECA452 or 10 ug/mL Rat IgM isotype control for 30 min at 4° C.and subsequently with 20 ug/mL of anti-Rat IgM-FITC for 30 min at 4° C.The flow cytometric results show an increase in HECA452 epitopeexpression on the cell surface after enforced fucosylation.

Example 6: In Vivo Fucosylation of Pulmonary Stem Cells

Pulmonary stem cells were treated with 60 mU/mL of FT-VI, 1 mMGDP-fucose or treated with buffer alone (HBSS, 0.1% human serum albumin)for 1 hour at 37° C. Cells were incubated with 10 ug/mL of HECA452 or 10ug/mL Rat IgM isotype control for 30 min at 4° C. and subsequently with20 ug/mL of anti-Rat IgM-FITC for 30 min at 4° C. The flow cytometricresults show an increase in HECA452 epitope expression on the cellsurface after forced fucosylation.

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A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of administering a population ofmodified cells comprising fucosylated CD44 structures that bindE-selectin to a subject comprising the steps of: (1) contacting apopulation of α(2,3)-sialylated CD44-expressing cells in vitro with apurified α-1,3-fucosyltransferase in a physiologically acceptablesolution comprising a fucose donor without the input of divalent metalco-factors, thereby forming the population of modified cells comprisingfucosylated CD44 structures that bind E-selectin, wherein the purifiedα-1,3-fucosyltransferase is capable of transferring 1.0 μmole of fucoseto an acceptor per minute at pH 6.5 at 37° C. in a physiologicallyacceptable solution comprising a fucose donor, and wherein thepopulation of modified cells has a viability of at least 70% at 24 hoursafter the contacting with the α-1,3-fucosyltransferase; and (2)administering the population of modified cells to the subject.
 2. Themethod of claim 1, wherein the population of modified cells is apopulation of stem cells or a population of differentiated cells.
 3. Themethod of claim 1, wherein the population of modified cells is apopulation of hematopoietic stem cells, a population of mesenchymal stemcells, a population of tissue stem/progenitor cells, a population ofumbilical cord stem cells, or a population of embryonic stem cells. 4.The method of claim 1, wherein the population of modified cells binds toone or more of E-selectin and L-selectin.
 5. The method of claim 1,wherein the population of modified cells is a population of lymphocyteor leukocyte cells.
 6. The method of claim 1, wherein the population ofmodified cells has a viability of at least 80% at 12 hours after thecontacting with the α-1,3-fucosyltransferase.
 7. The method of claim 1,wherein the population of modified cells has a viability of at least 80%at 24 hours after the contacting with the α-1,3-fucosyltransferase. 8.The method of claim 1, wherein the population of modified cells has aviability of at least 90% at 12 hours after the contacting with theα-1,3-fucosyltransferase.
 9. The method of claim 1, wherein thepopulation of modified cells has a viability of at least 90% at 24 hoursafter the contacting with the α-1,3-fucosyltransferase.
 10. The methodof claim 1, wherein the population of modified cells is a population ofmesenchymal stem cells.
 11. The method of claim 1, wherein the subjectis a human.
 12. The method of claim 1, wherein the subject is a mouse.13. The method of claim 1, wherein the population of modified cells isadministered allogeneically or autogeneically.
 14. The method of claim1, wherein the population of modified cells is administeredintravascularly.
 15. The method of claim 1, wherein the population ofmodified cells is administered intravenously.
 16. The method of claim 1,wherein the population of modified cells is effective to home to sitesof one or more of E-selectin and L-selectin expression in the subject.17. The method of claim 1, wherein the population of modified cells iseffective to home to the bone marrow of the subject.
 18. The method ofclaim 1, wherein the population of cells is also contacted with apurified sialyltransferase in a physiologically acceptable solutioncomprising a sialic acid donor without the input of divalent metalco-factors.
 19. The method of claim 1, wherein the population of cellscomprises CD34⁻ cells.
 20. The method of claim 1, wherein the populationof cells comprises CD34⁺ cells.